Regulation of tissue-specific expression during cellular development and differentiation in mammals is a complex process involving many different mechanisms. Numerous studies have addressed mechanisms regulating the actual initiation or activation of tissue-specific gene transcription, but activation must be preceded by potentiation of a locus, and much less is known about the mechanisms that regulate this process. There is mounting evidence that programmed changes in DNA methylation may be part of the potentiation mechanism; however, this has yet to be unequivocally demonstrated in an in vivo system. Many genes become demethylated specifically in the cell lineage in which they are expressed, but remain methlyated in non-expressing cells. This is the case for the autosomal phosphoglycerate kinase (Pgk2), which is expressed specifically in spermatogenic cells. This gene becomes demethylated several days prior to the activation of transcription. Recent results from this laboratory indicate that this demethylation event, which is gene-developmental-stage, and cell- type specific, is regulated by a cis-acting signal in the promoter region of the Pgk2 gene. The applicant's long-term goal is to conduct experiments to directly test the hypothesis that demethylation of the 5' portion of this gene is a prerequisite for transcriptional activation, using a novel, whole animal transgenic system developed for this purpose. However, to confirm the feasibility of this approach, the applicant must first demonstrate that this transgene system accurately mimicks the changes in DNA methylation that precede activation of tissue-specific transcription at the endogenous Pgk2 locus. Secondly, the applicant must demonstrate that different protein-DNA interactions regulate the demethylation and subsequent transcriptional activation processes, so that he can eventually disrupt the former in order to assess the effect on the latter. The aims of this R03 application are to conduct these preliminary experiments with the goal of obtaining results that will form the basis for a future R01 application to unequivocally establish, in an in vivo system, the role of DNA methylation in regulating tissue- specific gene expression.